Endotoxin is a lipopolysaccharide present in a cell wall of a Gram-negative bacterium and is the most typical pyrogen. If a transfusion, a medicine for injection, blood or the like contaminated with the endotoxin enters into a human body, the endotoxin may induce severe side effects such as fever and shock. Therefore, it is required to manage the above-mentioned medicines so that they are not contaminated with endotoxin.
By the way, a hemocyte extract of limulus (hereinafter, also referred to as “limulus amoebocyte lysate (LAL)”) contains serine protease that is an enzyme activated by endotoxin. When LAL reacts with endotoxin, a coagulogen present in LAL is hydrolyzed into a coagulin by an enzyme cascade by the serine protease activated according to the amount of endotoxin, and the coagulin is associated to form an insoluble gel. With the use of this property of LAL, it is possible to detect endotoxin with a high sensitivity.
Furthermore, β-D-glucan is a polysaccharide that constitutes a cell membrane characteristic of a fungus. Measurement of β-D-glucan is effective, for example, for screening of infectious diseases due to a variety of fungi including not only fungi that are frequently observed in general clinical practices, such as Candida, Aspergillus, and Cryptococcus, but also rare fungi.
Also in the measurement of β-D-glucan, by using the property of the limulus amoebocyte lysate to coagulate (coagulate to form a gel) by β-D-glucan, the β-D-glucan can be detected with a high sensitivity.
A method for detection or concentration measurement of a physiologically active substance of biological origin (hereinafter, also referred to as a “predetermined physiologically active substance”) such as endotoxin and β-D-glucan, which can be detected by a limulus amoebocyte lysate, includes a semi-quantitative gelation method which includes: by allowing a liquid mixture obtained by mixing a sample to be subjected to detection or concentration measurement of the predetermined physiologically active substance (hereinafter, also simply referred to as “measurement of the predetermined physiologically active substance”) with a reagent produced based on LAL (LAL reagent) to stand still; inverting the container after a lapse of a predetermined time; and determining whether or not the sample is gelled based on the presence or absence of dipping of the sample so as to examine whether or not the sample contains endotoxin of a certain concentration or more. Examples of the method include a turbidimetric method including analyzing a sample by measuring over time the turbidity of the sample caused by gel formation by a reaction between LAL reagent and the predetermined physiologically active substance, a colorimetric method using a synthetic substrate that is hydrolyzed by an enzyme cascade to develop a color, and other methods.
In the case where a predetermined physiologically active substance is measured by the above-mentioned turbidimetric method, a liquid mixture of a measurement sample and an LAL reagent is produced in a dry-heat-sterilized glass measurement cell. Then, gelation of the liquid mixture is optically measured from the outside. However, the turbidimetric method may require a very long time for gelation of the liquid mixture particularly in a sample with a low concentration of the predetermined physiologically active substance. To solve the problem, a method that can measure the predetermined physiologically active substance in a short time has been required. There has been proposed a laser light scattering particle counting method capable of forming gel fine particles by stirring a liquid mixture of a measurement sample and an LAL reagent using, for example, a magnetic stirring bar, and measuring the presence of the predetermined physiologically active substance in the sample in a short time based on the intensity of laser light scattered by the gel particles or based on the intensity of light transmitted through the liquid mixture.
The LAL reagent is produced by using limulus amoebocyte lysate as a principal raw material. Therefore, in production, endotoxin or β-D-glucan may enter with a certain probability, and the resultant product may be a waste that cannot be used as a reagent. Furthermore, since the measurement methods of any of endotoxin and β-D-glucan use a limulus amoebocyte lysate that is a limited resource as a raw material of the measurement reagent, it is necessary to reduce the amount of use of the reagent, or to reduce the production loss in producing the reagent.
As an attempt to reduce the amount of use of the reagent, not only simply reducing the volume of samples, but also enhancing the measurement sensitivity to consequently reduce the number of measurement times is effective. Meanwhile, for reducing the loss in production, it is important to suppress contamination with endotoxin or β-D-glucan. In addition, it should also be considered to remove endotoxin or β-D-glucan from a raw material that becomes unusable as a reagent during production and to reuse it as an auxiliary additive of the reagent. When the raw material is used as an auxiliary additive of the reagent, the use to be thought includes use as a coagulogen raw material that plays a role as an agent of adjusting the viscosity of a reagent, or a central role in gelation and aggregation.
However, it is impossible to remove an enzyme group, which has already been activated, only by removing endotoxin in the raw material that is contaminated with, for example, endotoxin and that cannot be used. A method for manufacturing a raw material, in which only an enzymatic activity in the raw material is inactivated while maintaining the function of coagulogen, that is, the function of being hydrolyzed by an activated clotting enzyme and converted into a coagulin to cause gelation and an aggregation reaction, has not been reported.